We have previously shown that NCB-20, a clonal hybrid of neuroblastoma x fetal Chinese hamster brain cell, expresses serotonin pre-synaptic components. These include a 5-HT uptake system and a specific binding pre-synaptic components. These include a 5-HT uptake system and a specific binding sites for imipramine which inhibits the uptake of 5-HT in a competitive manner. Imipramine and related tricyclic antidepressants also activate phosphoinositide hydrolysis by phospholipase C. These effects were nonadditive to those produced by stimulation of muscarinic cholinergic receptors with carbachol and can be desensitized by prestimulation with carbachol. In the present study, we have characterized the post-synaptic cyclase, using new specific agonists and antagonists for various classes of 5-HT receptors. Among various agonists examined, only 5- hydroxytryptamine (5-HT), 5-methoxytryptamine (5-MeOT) and the 5- HT3 agonist 2-methyl-5-hydroxytryptamine (2-Me-5-HT) increased basal accumulation of cyclic AMP by about 100% with an EC50 of about 0.5 muM, 1 muM and 10 muM, respectively. Moreover, we found no effect with the putative agonists for 5-H1A receptor: i.e., hydroxy-8-N, N-propylamine-2-tetraline (8-OH-DPAT), ipsapirone, buspirone or for 5-HT 1B receptor: 1-(3-trifluoromethylphenyl) piperazine (TFMPP), m-chlorophenylpipzine (m-CPP). The 5-HT effect on basal accumulation was blocked by metergoline but was relatively resistant to 5-HT receptor selective antagonists. A combination of forskolin with 10 muM of 5-HT. 5-MeOT or 2-Me-5-HT produced more than additive effect on cyclic AMP increase, while the agents acting on 5-HT 1A and 5-HT 1B receptors did not affect the response. Binding data suggest that these cells were virtually devoid of 5-HT 1A and 5-HT 1B receptor sites. Taken together, it appears that the 5-HT receptor present in this cell line cannot be simply classified as a 5-HT receptor previously described. In fact, this receptor possesses many characteristics of a novel "5- HT3-like" receptor.